The Triplet Expansion Diseases (TREDs) are a group of disorders that include Fragile X syndrome and Friedreich's ataxia. These disorders result from an expansion or increase in the number of triplet repeats in a tandem array of these triplets. The likelihood of expansion is related to the number of uninterrupted repeats. All currently known TREDs involve the triplets CGG, CAG or CTG. It is not clear whether this reflects ascertainment bias, or some fundamental property of these triplets. The mechanism of expansion is also unknown. To address these issues we are carrying out a systematic study of the stability of all 10 possible triplet repeats. We are also examining the DNA structure of these repeats, since we and others have shown that repeats associated with TREDs are all capable of forming intrastrand structures that might account for the triplet expansion. To date we have characterized the structures formed by (TGG)20 and (AGG)20 and shown that they form a series of tetraplexes similar to those we had previously found in other unstable repeats. This suggests that genes containing these repeats may be potential candidates for new triplet expansion defects. We have also shown that the ability to form such structures is a common property of very unstable sequences. We have also characterized a pair of unusual tetraplexes formed by one such sequence in mice. These tetraplexes involve novel tetrads containing a mixture of As and Gs. The ability of these sorts of tetrads to form suggests that the number of tetraplex-forming sequences in mammalian genomes is larger than previously thought. In collaboration with Dr Robert Nussbaum's laboratory at NHGRI, we have constructed trangenic mice containing variants of the 5' end of the human FMR1 gene. These variants differ in the number of CGG repeats found in the 5' UTR of the gene and correspond to normal and carrier alleles for Fragile X syndrome. No evidence of triplet expansion was seen in the offspring of these mice, indicating that while CGG repeats are necessary for expansion, they are not sufficient. Work is in progress using these mice to examine some of the other factors that might be important. Friedreich's ataxia results from an intronic expansion of the triplet GAA in the frataxin gene, which results in reduced frataxin mRNA levels. Dr Grabczyk has shown that GAA-repeats are intrinsically capable of attenuating transcription in a length dependent manner. He has shown that the molecular basis of this effect is the formation of a persistent RNA:DNA hybrid. Preliminary evidence suggests that formation of this hybrid is facilitated by transcription-driven triple-helix formation. This represents a novel, but potentially widespread, mechanism for transcriptional regulation.